DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

<p>Abstract</p> <p>Background</p> <p>Boron neutron capture reaction (BNCR) is based on irradiation of tumors after accumulation of boron compound. ¹⁰B captures neutrons and produces an alpha (⁴He) particle and a recoiled lithium nucleus (⁷Li). These particles have the characteristics of high linear energy transfer (LET) radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells.</p> <p>Methods</p> <p>Chinese hamster ovary (CHO-K1) cells and a DNA double-strand break (DSB) repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells), were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX) and 53BP1 foci using immunofluorescence intensity.</p> <p>Results</p> <p>Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness) estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity.</p> <p>Conclusion</p> <p>Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have an advantage over gamma-ray sensitive cells in BNCR.</p

In vitro characterization of cells derived from chordoma cell line U-CH1 following treatment with X-rays, heavy ions and chemotherapeutic drugs

<p>Abstract</p> <p>Background</p> <p>Chordoma, a rare cancer, is usually treated with surgery and/or radiation. However, very limited characterizations of chordoma cells are available due to a minimal availability (only two lines validated by now) and the extremely long doubling time. In order to overcome this situation, we successfully derived a cell line with a shorter doubling time from the first validated chordoma line U-CH1 and obtained invaluable cell biological data.</p> <p>Method</p> <p>After isolating a subpopulation of U-CH1 cells with a short doubling time (U-CH1-N), cell growth, cell cycle distribution, DNA content, chromosome number, p53 status, and cell survival were examined after exposure to X-rays, heavy ions, camptothecin, mitomycin C, cisplatin and bleocin. These data were compared with those of HeLa (cervical cancer) and U87-MG (glioblastoma) cells.</p> <p>Results</p> <p>The cell doubling times for HeLa, U87-MG and U-CH1-N were approximately 18 h, 24 h and 3 days respectively. Heavy ion irradiation resulted in more efficient cell killing than x-rays in all three cell lines. Relative biological effectiveness (RBE) at 10% survival for U-CH1-N was about 2.45 for 70 keV/μm carbon and 3.86 for 200 keV/μm iron ions. Of the four chemicals, bleocin showed the most marked cytotoxic effect on U-CH1-N.</p> <p>Conclusion</p> <p>Our data provide the first comprehensive cellular characterization using cells of chordoma origin and furnish the biological basis for successful clinical results of chordoma treatment by heavy ions.</p

Selective Effects of Emi1 Depletion in Cancer

Background: To improve the effectiveness of chemo- and radiotherapy only in cancer tissue is important for avoiding side effects. Results: Emi1 depletion enhanced the sensitivity of anticancer reagents and X-ray irradiation in cancer cells. Conclusion: Emi1 siRNA would be a useful new modality for enhancing the effect of chemo- and radiotherapy in various tumors. Significance: This work provides new insights regarding synergistic effect of Emi1 knockdown in combination therapies

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism

Repair of DNA damage induced by accelerated heavy ions - A mini review

Increasing use of heavy ions for cancer therapy and concerns from exposure to heavy charged particles in space necessitate the study of the basic biological mechanisms associated with exposure to heavy ions. Since the most critical damage induced by ionizing radiation is DNA double strand break (DSB), this review focuses on DSBs induced by heavy ions and their repair processes. Compared with X- or gamma-rays, high linear energy transfer (LET) heavy ion radiation induces more complex DNA damage, categorized into DSBs and non-DSB oxidative clustered DNA lesions (OCDL). This complexity makes the DNA repair process more difficult, partially due to retarded enzymatic activities, leading to increased chromosome aberrations and cell death. In general, the repair process following heavy ion exposure is LET-dependent, but with non- homologous end- joining (NHEJ) defective cells, this trend is less emphasized. The variation in cell survival levels throughout the cell cycle is less prominent in cells exposed to high LET heavy ions as compared with low LET, but this mechanism has not been well-understood until recently. Involvement of several DSB repair proteins is suggested to underlie this interesting phenomenon. Recent improvements in radiation induced foci (RIF) studies combined with high LET heavy ion exposure could provide a useful opportunity for more in depth study of DSB repair processes. Accelerated heavy ions have become valuable tools to investigate the molecular mechanisms underlying repair of DNA DSBs, the most crucial form of DNA damage induced by radiation and various chemotherapeutic agents

Strategies to Enhance Radiosensitivity to Heavy Ion Radiation Therapy

Heavy ion radiation therapy has been increasingly used due to several advantages over low linear energy transfer (LET) photon therapy, but further improvement of its therapeutic efficacy would be necessary. In this review, we summarize effective radiosensitizers for heavy ion radiation therapy and mechanisms associated with the radiosensitization. High LET heavy ions induce more complex and clustered DNA damage than low LET radiation. Inhibition of homologous recombimation repair or nonhomologous end rejoining and dysfunctional cell cycle checkpoint have been reported to sensitize cancer cells to heavy ions. Radiosenstizing agents, including DNA damage response inhibitors, Hsp90 inhibitors, histone acetylase inhibitors, and nanomaterials have been found to enhance cell killing by heavy ion irradiation through disrupted DNA damage response, cell cycle arrest, or other cellular processes. The use of these radiosensitizers could be a promising strategy to enhance the efficacy of heavy ion radiation therapy